Microvascular thrombosis and inflammation are interrelated in a variety of illnesses including sepsis, the body's inflammatory response to an infection. Sepsis is the most common admission diagnosis in intensive care units in the VA system and the leading cause of death in adult intensive care units in the United States. Veterans with sepsis and organ dysfunction have >50% mortality and microvascular thrombosis is implicated in organ dysfunction in sepsis. Thus, understanding the mechanisms responsible for microvascular thrombosis has major clinical significance to the VA health care system. Platelets are increasingly recognized to be important mediators of the inflammatory response, in addition to their well-established role in hemostasis and thrombosis. Recent work from this laboratory demonstrates that endotoxin, a major component of the cell wall of gram-negative bacteria, promotes microvascular platelet thrombus formation in vivo via toll-like receptor 4 (TLR4) on platelets. Endotoxin and platelet TLR4 promote release of histones, nuclear proteins that may also link inflammation, platelet activation and thrombosis. Our preliminary data show that histones promote microvascular thrombosis, they activate platelets and they induce endothelial cells to release von Willebrand factor (VWF). This proposal will address the mechanisms by which platelet TLR4 and histones promote microvascular thrombosis. The central hypothesis is that platelet TLR4 mediates microvascular thrombosis in endotoxemia via microparticles and histone-induced VWF release. The proposal will utilize in vivo and in vitro techniques; in vivo it will utilize two intravital microscopy modls well established in our lab and others. These models allow assessment of the kinetics of microvascular thrombosis and platelet- endothelial adhesive interactions in real-time. In vitro experiments involve assessment of platelet adhesion to cultured endothelial cells and immobilized substrates under flow and platelet aggregation. Two aims are proposed: Aim 1 will determine the mechanisms by which platelet TLR4 promotes microvascular thrombosis in endotoxemia. Aim 2 will determine the mechanisms by which histones promote microvascular thrombosis. Completion of the proposed experiments will broaden understanding of the links between inflammation and microvascular thrombosis in sepsis, and will provide the basis for future work aimed at preventing microvascular thrombosis in inflammation. The long-term goal is to develop optimal therapeutic approaches for patients with sepsis and other inflammatory diseases.